Gross chromosomal rearrangements (GCRs) have been identified as mutations that underlie different inherited genetic diseases, as mutations that drive the development of cancer and as loss of heterozygosity events that uncover recessive mutations in tumor suppressor genes. GCRs also result from the increased genome instability is seen in many cancers and may reflect a type of mutator phenotype. Inherited cancer susceptibility syndromes have been identified that are associated with increased spontaneous or DNA damage-induced genome instability, although whether the GCRs that arise drive the development of cancer is less clear. While genome instability is being intensely studied, our knowledge of the pathways and mechanisms that prevent genome instability remains limited. Understanding these mechanisms and pathways will impact on human health for several reasons: 1) Identifying the genes that suppress genome instability will provide new candidate tumor suppressor genes for investigation and candidate genes in which polymorphisms may interact with environmental agents; and 2) Many chemotherapeutic agents damage DNA and understanding how damage interacts with the pathways that suppress genome instability could lead to improvements in the efficacy of these agents as well as the development of new therapeutic approaches. In the proposed studies, Saccharomyces cerevisiae will be used as a model system to identify the genes and pathways that act to suppress GCRs. Key long term goals of these studies are to identify the types of metabolic errors, chromosomal features and mechanisms that contribute to genome instability and identify candidate genes in which defects might cause genome instability in cancer cells. The proposed work will build on insights obtained using previously developed quantitative systems for studying the formation of GCRs in S. cerevisiae. The following experimental approaches will be pursued: 1) New GCR assays and robust methods for mapping the structures of GCRs will be developed; 2) High throughput genetic analysis of a bioinformatics- derived set of enriched candidate genes will be performed using different GCR assays to identify the genes and pathways that suppress specific kinds of GCRs; 3) Genetic studies will be performed to determine the mechanisms that suppress GCRs mediated by duplicated sequences like segmental duplications; 4) The mechanistic features of some of the pathways that suppress GCRs will be investigated, initially focusing on the study of Rpa, Esc2, chromatin remodeling factors and the suppression of homeologous recombination; 5) Biochemical studies of homeologous recombination and break-induced replication will be performed to better understand how these pathways are regulated to prevent the formation of GCRs; and 6) Mouse and Human homologues of the S. cerevisiae genome instability genes will be identified to extend the study of genome instability to mouse and, ultimately, human systems. The results of these studies will be a comprehensive picture of the pathways and mechanisms that prevent GCRs.